Methods and apparatus for isolating platelets from blood

ABSTRACT

A platelet collection device comprising a centrifugal spin-separator container with a cavity having a longitudinal inner surface. A float in the cavity has a base, a platelet collection surface above the base, an outer surface. The float density is below the density of erythrocytes and above the density of plasma. The platelet collection surface has a position on the float which places it below the level of platelets when the float is suspended in separated blood. During centrifugation, a layer of platelets or buffy coat collects closely adjacent the platelet collection surface. Platelets are then removed from the platelet collection surface. Movement of a float having a density greater than whole blood through the sedimenting erythrocytes releases entrapped platelets, increasing the platelet yield.

BACKGROUND

1. Field

The present invention concerns apparatuses and methods for rapidfractionation of blood into erythrocyte, plasma and platelet fractions.Each fraction may be put to use or returned to the blood donor. Usefulhigh concentration platelet fractions have platelet concentrations inexcess of two times the concentration in anti-coagulated whole bloodbefore processing of greater than 2×10⁶ platelet/μL. The invention hasparticular value for rapid preparation of autologous concentratedplatelet fractions to help or speed healing.

2. Description of the Prior Art

Blood may be fractionated and the different fractions of the blood usedfor different medical needs. For instance, anemia (low erythrocytelevels) may be treated with infusions of erythrocytes. Thrombocytopenia(low thrombocyte (platelet) levels) may be treated with infusions ofplatelet concentrate.

Under the influence of gravity or centrifugal force, blood spontaneouslysediments into three layers. At equilibrium the top, low-density layeris a straw-colored clear fluid called plasma. Plasma is a water solutionof salts, metabolites, peptides, and many proteins ranging from small(insulin) to very large (complement components). Plasma per se haslimited use in medicine but may be further fractionated to yieldproteins used, for instance, to treat hemophilia (factor VIII) or as ahemostatic agent (fibrinogen).

The bottom, high-density layer is a deep red viscous fluid comprisinganuclear red blood cells (erythrocytes) specialized for oxygentransport. The red color is imparted by a high concentration of chelatediron or heme that is responsible for the erythrocytes high specificgravity. Packed erythrocytes, matched for blood type, are useful fortreatment of anemia caused by, e.g., bleeding. The relative volume ofwhole blood that consists of erythrocytes is called the hematocrit, andin normal human beings can range from about 38% to about 54%.

The intermediate layer is the smallest, appearing as a thin white bandon top the erythrocyte layer and below the plasma, and is called thebuffy coat. The buffy coat itself has two major components, nucleatedleukocytes (white blood cells) and anuclear smaller bodies calledplatelets (or thrombocytes). Leukocytes confer immunity and contributeto debris scavenging. Platelets seal ruptures in the blood vessels tostop bleeding and deliver growth and wound healing factors to the woundsite.

Extraction of Platelets

Extraction of platelets from whole blood has been reviewed (Pietersz2000). In transfusion medicine the intention is to transfuse eachpatient only with the component that is needed, so the aim of bloodcenters is to manufacture blood components as pure as possible, that iswith the least contaminating cells. Platelets are the most difficult toisolate and purify. Based on data from Pietersz (2000), even underoptimal conditions of centrifugation (long time at low speed), asignificant fraction of platelets remain trapped within the sedimentederythrocytes.

Through the years centrifugation methods have been developed to separatethe platelets from red blood cells, white blood cells and plasma. Thesemethods separate the components both in plastic bag systems and inapheresis devices, and more recently in specialized apparatuses.Historically most platelet concentrates have been harvested from donorsand used to treat thrombocytopenia, i.e., allogenically. More recentlythe platelet concentrates have been used to promote wound healing, andthe use of autologous platelet concentrates (sequestration of plateletsfor treatment of the platelet donor) has grown.

The sedimentation of the various blood cells and plasma is based on thedifferent specific gravity of the cells and the viscosity of the medium.This may be accelerated by centrifugation according approximately to theSvedberg equation:V=((2/9)ω² R(d _(cells) −d _(plasma))r ²)/η_(t)where

-   V=sedimentation velocity,-   ω=angular velocity of rotation,-   R=radial distance of the blood cells to the center of the rotor,-   d=specific gravity,-   r=radius of the blood cells,-   η_(t)=viscosity of the medium at a temperature of t° C.

Characteristics of blood components are shown in the table. DiameterSpecific gravity Component (μm) (g/ml) Deformability Adhesion Red cells5.4 1.100 +++ — Granulocytes 9.6 1.085 + ++ Lymphocytes 7.6 1.070 ± ±Monocytes 11.2 1.063 ± + Platelets 3.2 1.058 ± +++ Plasma NA 1.026 NA NAAdditive NA 1.007 NA NA solution

When sedimented to equilibrium, the component with the highest specificgravity (density) eventually sediments to the bottom, and the lightestrises to the top. But the rate at which the components sediment isgoverned roughly by the Svedberg equation; the sedimentation rate isproportional to the square of the size of the component. In other words,at first larger components such as white cells sediment much faster thansmaller components such as platelets; but eventually the layering ofcomponents is dominated by density.

Soft Spin Centrifugation

When whole blood is centrifuged at a low speed (up to 1,000 g) for ashort time (two to four minutes) white cells sediment faster than redcells and both sediment much faster than platelets (per Svedbergequation above). At higher speeds the same distribution is obtained in ashorter time. This produces layers of blood components that are notcleanly separated and consist of (1) plasma containing the majority ofthe suspended platelets and a minor amount of white cells and red cells,and (2) below that a thick layer of red cells mixed with the majority ofthe white cells and some platelets. The method of harvestingplatelet-rich plasma (PRP) from whole blood is based on this principle.The term “platelet-rich” is used for this component because most of theplatelets in the whole blood are in the plasma following slowcentrifugation so the concentration of platelets in the plasma hasincreased. Centrifugal sedimentation that takes the fractionation onlyas far as separation into packed erythrocytes and PRP is called a “softspin”. “Soft spin” is used herein to describe centrifugation conditionsunder which erythrocytes are sedimented but platelets remain insuspension. “Hard spin” is used herein to describe centrifugationconditions under which erythrocytes sediment and platelets sediment in alayer immediately above the layer of erythrocytes.

Two Spin Platelet Separation

Following a soft spin, the PRP can removed to a separate container fromthe erythrocyte layer, and in a second centrifugation step, the PRP maybe fractioned into platelet-poor plasma (PPP) and platelet concentrate(PC). In the second spin the platelets are usually centrifuged to apellet to be re-suspended later in a small amount of plasma.

In the most common method for PRP preparation, the centrifugation ofwhole blood for 2 to 4 min at 1,000 g to 2,500 g results in PRPcontaining the majority of the platelets. After the centrifugation of aunit (450 ml) of whole blood in a 3-bag system the PRP is transferred toan empty satellite bag and next given a hard spin to sediment theplatelets and yield substantially cell-free plasma. Most of the plateletpoor plasma (PPP) is removed except for about 50 ml and the pellet ofplatelets is loosened and mixed with this supernatant. Optionally onecan remove about all plasma and reconstitute with additive solution. Toallow aggregated platelets to recover the mixture is given a rest of oneto two hours before platelets are again re-suspended and then stored onan agitator.

It is believed that centrifugation can damage the platelets bysedimenting the platelets against a solid, non-physiological surface.The packing onto such a surface induces partial activation and may causephysiological damage, producing “distressed” platelets which partiallydisintegrate upon resuspension.

Hard Spin Centrifugation

If the centrifugation is continued at a low speed the white cells willsediment on top of the red cells whereas the platelets will remainsuspended in the plasma. Only after extended low speed centrifugationwill the platelets also sediment on top of the red cells.

Experiments with a blood processor (deWit, 1975) showed thatcentrifugation at a high speed (2,000 g-3,000 g) produces a similarpattern of cell separation in a shorter time. Initially the cellsseparate according to size, i.e., white cells sediment faster than redcells and platelets remain in the plasma. Soon the red cells get‘packed’ on each other squeezing out plasma and white cells. Because oftheir lower density, white cells and platelets are pushed upwards to theinterface of red cells and plasma whereas the platelets in the upperplasma layer will sediment on top of this interface, provided thecentrifugal force is sufficiently high and sedimentation time issufficiently long. Plasma, platelets, white cells and red cells willfinally be layered according to their density. Platelets sedimented atopa layer of red cells are less activated than those isolated by the “twospin” technique.

Platelet Yields and Centrifuge Speed

The so called “buffy coat” consists of the layers of platelets and whitecells (leukocytes) but is usually harvested along with the lower part ofthe plasma layer and the upper layer of the red cell mass. In thisapplication, all references to the platelet layer are intended to meanthe platelet layer if no leukocytes are present or to the buffy coatlayer when leucocytes are present mixed with the platelets.

The process and method of this invention can accomplish plateletisolation and collection with a wide range including both low and highcentrifugation forces. Effective separation does not require a high gcentrifugation; good results have been obtained with 600 g-1000 g or lowspeed centrifugation. High speed centrifugation refers to centrifugalforces greater than 2000 g. Experiments have shown that long (30-45 min)centrifugation at a force of about 700 g gives the most completeseparation of whole blood into components. Such long times are notconsidered to be practical and economical for intra-operative autologousapplications. For buffy coat separation one can spin 7 to 10 min atabout 3,000 g to enable separation of whole blood into cell-free plasma,a buffy coat containing 60-70% of the white cells and 70-80% of theplatelets, and red cells contaminated with approximately 30% of thewhite cells and 10-20% of the platelets.

Apheresis—Single Spin Platelet Separation

Specialized apparatuses have been invented to perform apheresis, theseparation of platelets from blood while reinfusing the other componentsinto the donor. This permits donors to give more platelets than possiblewith the two-step centrifugation because loss of erythrocytes limits thevolume of blood that blood donors may give. Typically, a two to threehour apheresis procedure will produce a platelet product containing3×10¹¹ platelets, equivalent to 6 or more conventional blood donations.

The first demonstration of a single-step method for preparation ofplatelet concentrates was reported more than 25 years ago (dewit 1975).In this first attempt complete separation between the different cellularcomponents could not be achieved, at least not in one step because ofconsiderable overlap in the presence of platelets, leukocytes anderythrocytes in the fractions collected after different centrifugationtimes and speed. Many improved apheresis methods and devices have beendeveloped and are described in cited patents.

In apheresis methods drawn blood is immediately mixed with ananticoagulant, centrifuged (Haemonetics, Baxter CS 3000 and Amicus, CobeSpectra, Fresenius AS 104, AS 204), and separated into componentsaccording to density. The buffy coat is recognized by eye or by opticalsensors and the platelet-rich layer is directed to a separate bag.Software of the various manufacturers has been adjusted to manufactureplatelet concentrates without white cell contamination, some requiringadditional filtration after the platelet harvest, others having specialtechniques or tools built into the apheresis systems.

Leukoreduction

The PC's resulting from both laboratory two spin processing andapheresis methods contain donor leukocytes. It was shown the white cellsnegatively affect platelet storage and may induce adverse effects aftertransfusion due to cytokine formation. Removal of leukocytes(leukoreduction) from PRP and PC is a major problem because non-selfleukocytes (allogeneic leukocytes) and the cytokines they produce cancause a violent reaction by the recipient's leukocytes. In 1999 the FDABlood Product Advisory Committee recommended routine leukoreduction ofall non-leukocytes components in the U.S. (Holme 2000). Therefore, muchof the prior art focuses on leukoreduction of platelet concentratesbecause non-autologous leukocytes excite deleterious immune reactions.Since the process of this invention provides a convenient way to quicklyharvest autologous platelets from the patient's blood, immune reactionsare not a risk, and the presence of leukocytes is of little or noconcern.

Autologous Platelets

Autologous platelets have been shown to have advantages in comparisonwith allogeneic platelets. Concerns about disease transmission andimmunogenic reactions, which are associated with allogeneic orxenogeneic preparation, are minimized. The fact that an autologouspreparation is prepared at the time of surgery reduces the risksassociated with mislabeling a sample, which might occur through alaboratory system. The use of autologous platelets obviates therequirement for time-consuming screening tests. Platelet activation hasless time to develop. Unlike stored platelets which become partiallyactivated, the activation status of autologous platelets, when firstproduced, was found to be similar to that in the original whole blood(Crawther 2000).

Platelets may be used as an adjunct for wound healing. Knightondescribes applying autologous platelet releasate to wounds to enhancehealing (Knighton 1986). More recent studies use platelets themselves.Marx describes platelet preparations that dramatically accelerate bonehealing following dental implant procedures (Marx 1998). Otherresearchers make similar claims for other medical procedures, forinstance, treatment of macular holes (Gehring 1999), improved healing incosmetic surgery (Man 2001), and use for hemostasis (Oz 1992).

In recent years devices originally invented to wash erythrocytes fromshed blood (autotransfusion devices) have been adapted to permitseparation of autologous platelets, usually intraoperatively. Thisprocedure has the important advantage that autologous leukocytes causeno reaction from patient leukocytes because they are self leukocytes, soremoval of leukocytes from PC's is no longer important. For example,sequestration of PRP reduces allogeneic transfusion in cardiac surgery(Stover 2000). Autotransfusion devices from a variety of manufacturers(e.g., ElectroMedics 500) can be used to make autologous plateletpreparations with high platelet concentrations.

The autotransfusion equipment used to make autologous plateletconcentrates requires a skilled operator and considerable time andexpense. Most devices require a large prime volume of blood. TheElectroMedics 500 withdraws 400 to 450 ml of autologous whole bloodthrough a central venous catheter placed during surgery. As it withdrawsthe blood the separator adds citrate phosphate dextrose (CPD) to achieveanticoagulation. The blood is then centrifuged into its three basiccomponents. The red blood cell layer forms at the lowest level, theplatelet concentrate layer in a middle level, and the PPP layer at thetop. The cell separator incrementally separates each layer, from theless dense to the more dense; therefore it separates PPP first (about200 ml) and PC second (about 70 ml), leaving the residual red bloodcells (about 180 ml). Once the PPP is removed, the centrifuge speed islowered to 2400 RPM to allow for a precise separation of the PC from thered blood cells. In fact, the platelets most recently synthesized, andtherefore of the greatest activity, are larger and mix with the upper 1mm of red blood cells, so that this layer is included in the PRP productimparting a red tint.

Recently devices have been introduced which are specifically designed tomake autologous platelet concentrates intraoperatively; for example theSmartPReP Autologous Platelet Concentrate System (Harvest AutologousHemobiologics, Norwell, Mass.). It requires 90 to 180 cc of blood versusthe 500 cc of blood used in most autotransfusion machines. In additiontwo other products are near market introduction, The PlasmaSeal device(PlasmaSeal, San Francisco, Calif.) and The Platelet ConcentrateCollection System (Implant Innovations, Inc., Palm Beach Gardens, Fla.).While these devices have somewhat reduced the cost and the timerequired, a skilled operator is required for the devices introduced tothe market to date. Therefore, there remains a need for simple and fastautomated methods and devices for making platelet concentrates.

SUMMARY OF THE INVENTION

The present invention is directed to methods and apparatuses for simpleand fast preparation of autologous platelet concentrates from wholeanti-coagulated blood.

This discussion includes numerous descriptions of events within thespinning rotor. Within the frame of reference of the rotor, the effectsof gravity are minimal compared with centrifugal force. Therefore withinthe rotor, “top” means the end of the tube closer to the axis and“bottom” means the end of the tube closer to the perimeter of the rotor.

Another aspect of the present invention is that platelets are notaggregated by pelleting against a surface.

A further aspect of the invention is the use of a float having a densityless than the density of the erythrocytes and greater than that of wholeblood which rises through the mixture as the erythrocyte sediment duringcentrifugation, gently disrupting the erythrocytes to free trappedplatelets, thus greatly increasing the platelet yield.

Another aspect of the present invention is that the apparatuses may becompletely automated and require no user intervention between, first,loading and actuating the device and, second, retrieving the plateletconcentrate.

Another aspect of the present invention is that different quantities ofblood may be processed by the same apparatus.

Another aspect of the present invention is that bloods of differenthematocrits and different plasma densities may be processed by the sameapparatus.

Another aspect of the present invention is that the concentration ofplatelets in the product may be varied by need.

Another aspect of the present invention is that the processing includesonly a single centrifugation step.

Another aspect of the present invention is that the processing is rapid.

The float collector blood platelet separation device of this inventioncomprises a centrifugal spin-separator container having a separationchamber cavity with a longitudinal inner surface. A float is positionedwithin the cavity, the float having a base, a platelet collectionsurface above the base, and an outer surface. The distance between theouter surface of the float and the inner surface of the cavity can be0.5 mm, preferably less than 0.2 mm and optimally less than 0.03 mm. Thefloat has a density less than the density of erythrocytes and greaterthan the density of plasma. The platelet collection surface has aposition on the float which places it immediately below the level ofplatelets when the float is suspended in fully separated blood. Thecavity can have a cylindrical inner surface and the float has acomplementary cylindrical outer surface.

In one embodiment, the device includes a flexible inner tube, and afloat is positioned within the flexible inner tube. The float has anouter surface in sealing engagement with the inner surface of theflexible tube in a neutral pressure condition, the sealing engagementpreventing movement of fluid between the outer surface of the float andthe inner surface of the flexible tube in the neutral pressurecondition. The outer surface of the float disengages from contact withthe inner surface of the flexible tube in an elevated pressurecondition, thus enabling movement of fluid between the outer surface ofthe float and the inner surface of the flexible tube in the elevatedpressure condition as well as free movement of the float within thetube. The float has a platelet receptor cavity positioned to be at theposition of platelets in separated blood after centrifugation. The floathas a channel communicating with the platelet receptor cavity forremoving separated platelets therefrom after centrifugation. In oneconfiguration, the float comprises a proximal segment having a distalsurface and a distal segment having a proximal surface opposed to thedistal surface, the distal surface and the proximal surfaces definingthe platelet receptor cavity. Preferably, the outer container includes aport for introducing blood into the inner tube at the beginning of aplatelet separation process and for removing platelets from the plateletcavity within the inner tube at the end of the platelet separationprocess. Optionally, the port includes a syringe coupling Luer lockingdevice. The outer container can have an inner surface for restrainingexpansion of the inner tube during centrifugation.

In a still further embodiment, the centrifugal spin-separator is asubstantially rigid tube, and the float comprises a proximal segmenthaving a distal surface, and a distal segment having a proximal surfaceopposed to the distal surface, the distal surface and the proximalsurfaces defining the platelet receptor cavity. This cavity has asurface which is a platelet collection surface. The outer surface of thefloat is preferably in sliding engagement with the inner surface of thecavity.

The term “platelet collection surface”, as used herein, is defined tomean a surface which provides support to the platelet or buffy coatlayer. Preferably, the platelet layer is not in direct contact with thesupport layer to protect the platelets, and optimally, the platelets aresedimented on a thin buffer or cushion layer of erythrocytes resting onthe platelet collection surface.

In another embodiment, a top surface of the float constitutes a plateletcollection surface. In this form, the device may include a plungerpositioned above the float and substantially axially concentric with thefloat and the cavity, the optional plunger having a cylindrical outersurface which is spaced from a complementary cylindrical inner surfaceof the tube. The space can be so small as to provide an effective liquidseal between the surfaces, or if the space is larger, at least one sealcan be provided between the outer surface of the plunger and the innersurface of the cavity, the seal being positioned in sealing engagementwith the outer and inner surfaces. Optionally, the bottom of the plungerhas a plasma expressing surface opposed to the platelet collectionsurface; and a fluid removal passageway extends through the plunger andthe plasma expressing surface into the platelet receptor cavity.Preferably, the top of the float includes a stop surface extending abovethe plasma collection surface.

The process of this invention for separating platelets from whole bloodwith the above devices comprises the steps of first introducing anamount of whole blood into the cavity, the amount of whole blood beingsufficient, following centrifugation, to elevate the float above thefloor of the separation chamber and position the platelet collectionsurface immediately below the level of platelets. The separation chamberis the cavity within which the blood is separated into erythrocyte,plasma and platelet (buffy coat) layers. The centrifugal spin-separatorcontainer is subjected to centrifugation forces in the axial directiontoward the distal end, whereby erythrocytes are caused to concentrate atthe distal end, plasma to collect toward the proximal end, and plateletsto collect on the platelet collection surface. Platelets are thenremoved from the platelet collection surface.

When the device includes a plunger positioned above the float andsubstantially axially concentric with the float and the cavity, processof this invention comprises the steps of introducing an amount of wholeblood into the cavity, the amount of whole blood being sufficient toposition the level of platelets following centrifugation at the positionof the platelet collection surface. The centrifugal spin-separatorcontainer is then subjected to centrifugation forces in the axialdirection toward the distal end, whereby blood cells are caused toconcentrate at the distal end, plasma to collect toward the proximalend, and platelets to collect closely adjacent the platelet collectionsurface. The plunger is then advanced in an axial direction against thetop of the plasma until the plasma expressing surface is positionedclosely adjacent the platelet collection surface and spaced aparttherefrom. A platelet extraction tube is extended through the fluidremoval passageway until the end thereof contacts the platelet layer,and a platelet concentrate is removed through the platelet extractiontube. Optionally, platelet poor plasma can be collected through theplatelet extraction tube into a syringe or other receptacle while theplunger is being depressed. Platelets can then be extracted into aseparate syringe or other receptacle.

Optionally, the device can lack a plunger arrangement. In thisembodiment, platelets are removed from the platelet collection surfacesuspended in a small volume of plasma retained after first removing avolume of platelet poor plasma from above the sedimented platelet layer.

With embodiments of the device wherein the top of the float includes astop surface positioned above the plasma collection surface, the plungeris advanced in an axial direction until the plasma expressing surfacecontacts the stop surface.

With devices having a float in a flexible tube, the process comprisesthe steps of introducing an amount of whole blood into the inner tube,the amount of whole blood being sufficient, following centrifugation, toelevate the float above the floor of the separation chamber and positionthe platelet collection surface immediately below the level ofplatelets. The tube is then subjected to centrifugation forces in theaxial direction toward the distal end, whereby blood cells are caused toconcentrate at the distal end, plasma to collect at the proximal end,and platelets to collect at a level closely adjacent the plateletcollection surface. Platelets are then removed from the annular plateletreceptor cavity.

When the top surface of the float constitutes the platelet collectionsurface, the device optionally includes a plunger positioned above thefloat and substantially axially concentric with the float and thecavity. The plunger has a cylindrical outer surface which is spaced fromthe inner surface of the cavity; the bottom of the plunger defining aplasma expressing surface opposed to a platelet collection surface. Afluid removal passageway extends through the plunger to the plasmaexpressing surface. With this embodiment, the process includes theadditional step of moving the plunger toward the float until the plasmaexpressing surface is closely adjacent the platelet layer, and plateletsare then removed through the fluid removal passageway. In thisembodiment, plasma is expressed through the fluid removal passageway asthe plunger is moved toward the float.

BRIEF DESCRIPTION OF DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a schematic cross-sectional drawing of a separation device ofthis invention.

FIG. 2 shows device of FIG. 1 wherein the plunger of the syringe iselevated, and the syringe barrel is filled with blood.

FIG. 3 shows device of FIG. 1 after the plunger is depressed to aposition forcing the blood into the inner tube.

FIG. 4 shows device of FIG. 1 after a portion of the blood has passedbetween float and the inner tube, filling the bottom of the inner tube.

FIG. 5 shows device of FIG. 1 after the blood has separated into theerythrocyte fraction within which the float rests, the plasma fractionabove the float, and the buffy coat or platelet layer in the receptorcavity.

FIG. 6 shows device of FIG. 1 after a fresh syringe has been connectedto the Luer port.

FIG. 7 shows device of FIG. 1 with the syringe plunger elevated afterdrawing platelets from the receptor cavity into the barrel of thesyringe.

FIG. 8 is an isometric view of an alternative float design for theseparation device of FIG. 1, the bottom of the float having ahemispherical shape.

FIG. 9 is a schematic cross-sectional view of a plunger-float embodimentof the invention.

FIG. 10 is a schematic cross-sectional view of the embodiment of FIG. 9after introduction of anticoagulated blood into the separation chamber.

FIG. 11 is a schematic cross-sectional view of the embodiment of FIG. 9after centrifugal separation of the blood into erythrocyte, plasma andplatelet layers.

FIG. 12 is a schematic cross-sectional view of the embodiment of FIG. 9after insertion of a syringe needle.

FIG. 13 is a schematic cross-sectional view of the embodiment of FIG. 9after depression of the plunger to a level which abuts the float stop.

FIG. 14 is a schematic diagram view of an alternate embodiment relatedto the embodiment of FIG. 9 showing the introduction of blood through aseparate fill port of the embodiment.

FIG. 15 is a schematic diagram of a still further alternate embodimentof a plunger-float device of this invention, including a flexiblesnorkel tube fixed to the cap.

FIG. 16 is a schematic cross-sectional view of a plunger-floatembodiment of the invention after centrifugation and before removal ofthe platelet layer.

DETAILED DESCRIPTION

This invention is a blood platelet separation device with severalembodiments. All of the embodiments comprise a centrifugalspin-separator container having a cavity with a longitudinal innersurface. A float is positioned within the cavity. The float has a baseand a platelet collection surface above the base. The float has an outersurface. In general, the distance between the outer surface of the floatand the inner surface of the cavity can be less than 0.5 mm, preferablyless than 0.2 mm and optimally less than 0.03 mm. For embodiments with aflexible tube, the surfaces can be in contact. The platelet collectionsurface has a position on the float which places it immediately belowthe level of platelets when the float is suspended in fully separatedblood.

Patient blood may be obtained by a phlebotomy needle or central veincannula or other whole blood collection means. The blood is immediatelymixed with anticoagulant, such as ACD-A or heparin.

FIG. 1 is a schematic cross-sectional drawing of a separation device ofthis invention. The blood platelet collection device of this embodimentcomprises a flexible inner tube 2 having an inner surface 4 and a float6 positioned within the flexible inner tube. The float has an outersurface 8 in sealing engagement with the inner surface of the flexibletube when the tube is under neutral pressure. In this condition, thesealing engagement prevents movement of fluid between the outer surfaceof the float and the inner surface of the flexible tube.

The outer surface 8 of the float 6 disengages from contact with theinner surface 4 of the flexible tube 2 when the pressure in the flexibletube is elevated under centrifugation. This enables movement of fluidbetween the outer surface of the float and the inner surface of flexibletube as well as free movement of the float within the tube.

The float has a platelet receptor cavity 10 with a platelet collectionsurface 16 in a position to immediately below the level of platelets inseparated blood following centrifugation. The float 6 has a plateletcollection channel 11 and a platelet withdrawal channel 12 communicatingwith the platelet receptor cavity 10 for removing separated plateletsafter centrifugation.

The float 6 comprises a proximal segment 13 having a distal surface 14and a distal segment 15 not having proximal surface 16 opposed to thedistal surface 14. The distal surface 14 and proximal surface 16 definethe platelet receptor cavity 10. The float 6 has a specific gravity thatis less then the specific gravity of erythrocytes and greater than thespecific gravity of plasma such that at equilibrium the buffy coatplatelet layer is sequestered between the upper and lower members of thefloat. For optimum platelet recovery, it is critical that the float risefrom the bottom of the tube as the erythrocytes sediment. This requiresthat the float have a density greater than whole blood.

The platelet collection device of this embodiment includes asubstantially inflexible outer container 18 enclosing the inner tube 2.The inner surface 22 of the outer container 18 limits expansion of theinner tube as the pressure in the inner tube 2 increases duringcentrifugation.

The outer container includes a port 20 for introducing blood into theinner tube at the beginning of the platelet separation process and forremoving platelets from the platelet receptor cavity 10 through channels11 and 12 at the end of the platelet separation process. The port can beprovided with a Luer lock device for coupling with a loading syringe andwith a platelet removal syringe.

Vent channel 17 vents air upward through channel 12 as blood isintroduced into the separation channel 19.

In this embodiment, the needle or small tube 23 is preferably fixed tothe Luer lock device 20. The tube 23 has an outer diameter which issmaller than the inner diameter of the channel 12 to enable it to slidefreely in the channel 12 as the float 6 rises during centrifugation.

The device of this invention can be used in a simple operation toproduce platelets. It involves the collection of blood containing ananticoagulant such as heparin, citrate or EDTA in a syringe; filling theseparation tubes with the anti-coagulated blood from the syringe;centrifugation to separate the blood into erythrocyte, plasma andplatelet buffy coat fractions; and removal of the platelets buffy coatfraction with another syringe.

The float can be made of two cones, the bases thereof optionallyconcave. The separation chamber cavity preferably has a concave bottomwhich mirrors the shape of the lower cone so that when the buoy is inits initial state, resting at the bottom of the cavity, there is a smallspace between the bottom of the lower buoy and the bottom of the cavity.The flexible tube 2 is preferably an elastomer sleeve having an innerdiameter which is smaller than the greatest outer diameter of the floatso that it holds the float firmly in place. The outer diameter of theflexible tube 2 is smaller than the inner diameter of the rigid cylinder18 so that a space exists between the inner tube and the rigid cylinder.Small particles such as smooth spheres, e.g., ball bearings, can beprovided in the space between the two cones to disperse platelets in theplatelet buffy coat layer. The channel 12 terminates slightly above thebase of float 6. A sterile vent 21 allows air to pass in and out of thedevice.

FIGS. 2-7 are sequential schematic cross-sectional drawings of thedevice of this invention at the different phases of the separationprocess.

FIG. 2 shows the plunger 24 of the syringe 26 elevated, and the syringebarrel 28 is filled with blood 30.

FIG. 3 shows the plunger 24 depressed to a position forcing the blood 30into the inner tube 2.

FIG. 4 shows the position after a portion of the blood 30 passes betweenfloat and the inner tube 2 during centrifugation, filling the bottom 32of the inner tube.

FIG. 5 shows the blood components after centrifugation for a sufficienttime to separate the blood components into the erythrocyte fraction 34in which the float 6 floats, the plasma fraction 36 above the float 6,and the buffy coat or platelet layer 38 in the receptor cavity 10. Uponcessation of centrifugation, the blood remains fractionated into itsthree components, and the position of these components remains the samerelative to the float. No longer under pressure produced by centrifugalforce, the elastomer sleeve 2 has shrunk away from rigid cylinder 18 andlocks the buoy in place.

Surprisingly, with the current invention, a much smaller fraction ofplatelets remain associated with the erythrocyte pack, making higheryields of sequestered platelets possible. The float rising from thebottom of the device as erythrocytes sediment fluidizes the erythrocytepack to release the platelets so they more readily rise to combine withthe buffy coat.

If resuspension particles are present in the platelet receptor, theentire device can be shaken or rotated so that the particles tumblearound within the space between the two cones, disrupting and mixing thebuffy coat into a homogeneous suspension. Alternatively, the plateletscan be re-suspended by jetting in and out of the platelet-containingcompartment with the collection syringe. Alternatively, an air bubblecan be trapped within or introduced into the platelet-containingcompartment, and the platelets can be re-suspended by shaking, invertingor rolling the device. The suspended buffy coat is then withdrawn thoughthe Lehr 20. The removed volume is displaced by air which enters thedevice through vent 21.

FIG. 6 shows the device with a fresh syringe 40 locked to the Luer port20.

FIG. 7 shows the syringe plunger 42 elevated after drawing platelets 46from above the platelet collection surface 16 in the receptor cavity 10into the barrel 44 of the syringe. The suspended platelet layer has beenwithdrawn through the Luer 20. The removed volume is replaced by airwhich enters the device through a sterile vent 21 and further into theplatelet receptor 10. The syringe 40 containing the platelets 46 is thenremoved for provision of the platelets to the physician treating apatient (not shown).

FIG. 8 is an isometric view of the float component 90 with a plateletreceptor 92, a vent channel 93 extending to the interior of thecollection tube 96, and a platelet drainage channel 94 extending fromthe platelet receptor 92 to the interior of collection tube 96. Thisembodiment has a hemi-spherical bottom 98. The cylinder preferably has aconcave bottom which mirrors the hemi-spherical bottom 98 so that whenthe buoy is in its initial state, resting near the bottom of thecylinder, space between the float and bottom are minimized. Theprojection 97 extending from the bottom of the hemispherical bottom 98insures that a space is maintained between the bottom of the lower buoyand the bottom of the cylinder to prevent vacuum sticking of the floatto the bottom of the tube.

FIG. 9 is a schematic cross-sectional view of a plunger-float embodimentof the invention. In this embodiment, an axially concentric float 100and plunger 102 are contained within the central cavity 103 of the rigidtube or cylinder 104 with a cap 106. The cap 106 has a vent hole 108 forpermitting movement of air into and out of the tube when blood is addedor platelets are removed. It includes a Luer port 110 for receiving aneedle or tube used for introducing blood into the cylinder and forremoving fluid from the cylinder.

The float 100 has a bottom surface 112 with a projecting spacer 113which rests on the bottom 114 of the tube before anti-coagulated bloodis introduced into the separator. The float has an upper surface 115which is positioned to be immediately below the layer of platelets inseparated blood. The upper structure of the float includes a projection116, the top edge 118 of which acts as a stop to limit downward movementof the plunger 102 during the process. A platelet collection channel 120is positioned in the center of the float. Platelet drainage channels 122extend from the level of the surface 115 to the interior of the plateletcollection channel 120.

The float 100 has a density less than separated erythrocytes and greaterthan plasma so that it will float on the erythrocyte layer at a levelwhich places the platelet collection surface 115 immediately below theplatelet layer when the blood is separated into its components. Foroptimum platelet recovery, it is critical that the float rise from thebottom of the tube as the erythrocytes sediment. This requires that thefloat have a density greater than whole blood.

The plunger 102 optionally can have an outer surface 124 which is spacedfrom the inner surface 126 of the tube 104 or in sliding engagementtherewith. In the illustrated embodiment, seals 128 and 130 which can beO-rings are provided to prevent escape of liquid between the float andtube surfaces when the plunger 102 is moved toward the float 100. If thetolerances between the outer surface 124 and the tube surface 126 aresufficiently small, no seal is required to prevent escape of liquidbetween the plunger and the tube when the plunger is moved toward thefloat and when the product is withdrawn.

The plunger has a bottom surface 132 and a fluid escape or snorkel tube134. When the plunger is moved downward toward the float, the pressureimparted by this bottom surface 132 expresses liquid below the plunger102 upward through the snorkel tube 134 into the cavity above theplunger.

The plunger is provided with a central channel 136 through which a tubeor needle is inserted to remove platelet-rich fluid from the spacebetween the bottom of the plunger and the top of the float.

While this embodiment is illustrated with an outer tube and a float andplunger with matching outer cylindrical shapes, it will be readilyapparent to a person skilled in the art that the outer container canhave any internal shape which matches the dimensions of the float andplunger such as a cavity with a square or other polygonal shape combinedwith a float and plunger with the corresponding outer polygonal shape.The cylindrical configuration is advantageous.

FIG. 10 is a schematic cross-sectional view of the embodiment of FIG. 9after introduction of anti-coagulated blood 138 into the separationchamber 103 through a tube 140 from a syringe 142.

FIG. 11 is a schematic cross-sectional view of the embodiment of FIG. 9after centrifugal separation of the blood into erythrocyte 144, plasma146 and platelet 148 layers. The float 100 has risen to place theplatelet collection surface 115 immediately below the level of theplatelets 148.

FIG. 12 is a schematic cross-sectional view of the embodiment of FIG. 9after insertion of a syringe needle 150 of syringe 143 into the centralchannel 136 of plunger 102.

FIG. 13 is a schematic cross-sectional view of the embodiment of FIG. 9after depression of the plunger 102 by pressing the syringe 143downward, to a level which contacts its lower surface 132 with the stoptip 118 of the float 100. The plasma displaced by the plunger 102 hasbeen expressed through the snorkel tube 134.

Withdrawal of the piston 150 of the syringe 143 draws a platelet-richmixture from the platelet layer through the channels 122 and 120 (FIG.9) and upward through tube 150 into the syringe tube 145. The positionof the snorkel tube 134 above the liquid level provides for flow of airto fill the space created by removal of the platelet suspension.

FIG. 14 is a variation of the embodiment shown in FIG. 9, with theaddition of an optional port 151. This view shows blood 154 introducedthrough port 151 from syringe 142, flowing down channel 136 into theseparation chamber 103.

FIG. 15 is a schematic cross-sectional view of a plunger-floatembodiment of the invention. In this embodiment, an axially concentricfloat 160 and plunger 162 are contained within the separation chambercavity 163 of rigid tube or cylinder 164 with a cap 166. The float has aplatelet collection surface 165 which is positioned to be immediatelybelow the layer of platelets in separated blood. The cap 166 has a venthole 168 for permitting the escape of air from the tube when it blood isadded to its interior. It also has a Luer 170 which receives a needle ortube for introducing blood into the separation chamber 163 and anotherneedle or tube for removing fluid containing platelets from closelyadjacent the platelet collection surface 165 following centrifugation.

The float 160 has a bottom surface 172 which rests on the bottom 174 ofthe tube before anti-coagulated blood is introduced into the separator.The upper structure of the float includes a projection 176, the top edge178 of which acts as a stop to limit downward movement of the plunger162 during the process. A platelet collection channel 180 in the centerof the float communicates with platelet drainage channel 182 extendingfrom the level of the surface 165.

The float 160 has a density less than separated erythrocytes and greaterthan plasma so that it will float in the erythrocyte layer at a levelwhich places the platelet collection surface 165 immediately below theplatelet layer when the blood is separated into its components. Foroptimum platelet recovery, it is critical that the float rise from thebottom of the tube as the erythrocytes sediment. This requires that thefloat have a density greater than whole blood.

As the plunger 162 is depressed toward the float 160 aftercentrifugation, plasma rises through the flexible snorkel tube 188 intothe space 186 above the plunger 162. When platelets are removed by atube extending through the central channel 184 (inserted as shown inFIG. 13), air flows through the tube 188 from its inlet at the top ofthe tube (above the liquid level) to replace the liquid being removed.

The plunger is shown at its highest level to permit introducing amaximum amount of blood into the separation chamber, the maximum heightbeing limited by the top 190 of the tube 192 abutting the cap 166. Thisfull extension is permitted by the flexibility of the snorkel tube 188.

FIG. 16 is a schematic cross-sectional view of a plunger-floatembodiment of the invention after centrifugation and before removal ofthe platelet layer. In this embodiment, a float 200 is contained withinthe central cavity 202 of the rigid tube or cylinder 204 with a cap 206.The cap 206 has a vent hole 208 for permitting movement of air into andout of the tube when blood is added or platelets are removed. Itincludes a port 210 for receiving a needle or tube used for introducingblood into the cylinder and for removing fluid from the cylinder and aport 212 for receiving a syringe needle (not shown) for collectingplatelets following centrifugation.

The float 200 has a bottom surface 214 with a projecting spacer 216which rests on the bottom of the tube before anti-coagulated blood isintroduced into the separator. The float rises in the erythrocyte layerduring centrifugation. The float 200 has an upper surface 218 which ispositioned to be immediately below the layer of platelets in separatedblood. The upper structure of the float includes a projection 220 whichextends above the platelet or buffy coat layer.

The float 200 has a density less than separated erythrocytes and greaterthan plasma so that it will float in the erythrocyte layer at a levelwhich places the platelet collection surface 218 immediately below theplatelet layer when the blood is separated into its components. Foroptimum platelet recovery, it is critical that the float rise from thebottom of the tube as the erythrocytes sediment. This requires that thefloat have a density greater than whole blood.

The “Plungerless plunger” device of FIG. 16 is the simplest and cheapestto manufacture. The user can vary the platelet concentration factorsimply by removing more or less platelet poor plasma before resuspendingthe platelets. It requires more user attention and care to accuratelyremove the desired amount of platelet poor plasma. The parasol floatsystem of FIG. 1 and the plunger-float system of FIG. 9 provide betterreproducibility than the simple float embodiment of FIG. 16.

This invention is further illustrated by the following specific, butnon-limiting examples.

EXAMPLE 1 Parasol Float Device

A parasol design platelet concentrator device of the type depicted inFIG. 1 was constructed. The float was comprised of polyethylene andpolycarbonate in such proportion as to have an overall density of 1.06g/ml. The outer diameter of the float was 2.62 cm and its overall lengthwas 4.57 cm. The float together with two stainless steel balls 0.32 cmin diameter in the platelet receptor cavity was inserted into the sealedend of a flexible silicone rubber tube. The flexible tube had an innerdiameter of 2.54 cm, a wall thickness of 0.08 cm, and a sealed distalend. The flexible tube containing the float was housed within a rigidpolycarbonate tube with inner diameter of 2.86 cm and length 11.43 cm.The top of the flexible tube was folded over the top of the rigid tubeand a cap with a 7.62 cm tube 23 (see FIG. 1) was fitted over the foldedtop of the flexible tube with tube 23 engaging channel 12.

The device was filled with 30 ml of freshly drawn whole bloodanti-coagulated with CPDA-1. The device was centrifuged in an IEC CentraCL2 centrifuge for 30 minutes at 3000 rpm. Following centrifugation thetube was swirled vigorously to resuspend the platelets within theplatelet receptor cavity by the agitation induced by the stainless steelballs. Five cc concentrated platelets was removed from the plateletreceptor cavity through the platelet extraction tube (23).

Platelet counts were determined as follows: One half cc of this samplewas diluted with 10 cc of Isoton II isotonic diluent and centrifuged at500 g for 1.5 minutes. One half cc of this diluted sample was diluted inyet another 10 cc of Isoton II and particles larger than 3 fl counted ona Coulter Z-1 particle analyzer. This result was compared to the numberof particles in a similarly treated sample of whole blood. These smallparticles from treated samples represent the platelets. The sample ofconcentrated platelets contained 66% of the platelets present in theintroduced whole blood at a concentration 2.86 times that found in thewhole blood.

The “Parasol” device shown in FIG. 1 is most difficult and expensive tomanufacture, but is easiest to use. The erythrocyte concentration ismore variable with this product. This results from different plasmadensities, and hematocrit-dependant variability is present in the amountof displacement of fluid by contraction of the elastomeric sleeve duringdeceleration.

EXAMPLE 2 Plunger-Float Device with Snorkel

A platelet concentrator device of the type depicted FIG. 9 wasconstructed. The float was comprised of polyethylene and polycarbonatein such proportion as to have an overall density of 1.08 g/ml. The outerdiameter of the float was 2.535 cm and its overall length was 1.2 cm.The float was inserted into a rigid polycarbonate tube with an innerdiameter of 2.540 cm and length 11.43 cm. The bottom of the rigid tubewas sealed.

The device was filled with 25 cc of freshly drawn whole bloodanti-coagulated with CPDA-1. The device was centrifuged in an IEC CRU5000 centrifuge for 15 minutes at 1800 rpm. Following centrifugation theplunger was depressed by inserting a blunt hypodermic needle connectedto a 10 cc syringe through the central access port until it collidedwith the stop on the top of the float. The device was swirled vigorouslyto resuspend the platelets within the platelet receptor cavity afterwithdrawing 0.5 cc through the hypodermic needle (platelet extractiontube). An additional 3.5 cc concentrated platelets was removed from theplatelet receptor cavity through the hypodermic needle (plateletextraction tube).

One half cc of this sample was diluted with 10 cc of Isoton II isotonicdiluent and centrifuged at 500 g for 1.5 minutes. One half cc of thisdiluted sample was diluted in yet another 10 cc of Isoton II andparticles larger than 3 fl counted on a Coulter Z-1 particle analyzer.This result was compared to the number of particles in a similarlytreated sample of whole blood. These small particles from treatedsamples represent the platelets. The sample of concentrated plateletscontained 69% of the platelets present in the introduced whole blood ata concentration 4.30 times that found in the whole blood.

The “Plunger” device shown in FIG. 9 has advantage of being cheap tomanufacture and having less variability in percent erythrocytes in theproduct. The plunger-float combination provides a greater concentrationfactor because the volume between plunger and float can be smaller andstill accommodate the entire range of plasma densities while keeping thelevel of the buffy coat within the gap. Erythrocyte contamination isindependent of hematocrit.

EXAMPLE 3 Plunger-Float Device without Snorkel

A platelet concentrator device of the type depicted in FIG. 9 wasconstructed, except without the snorkel tube so that the only fluidcommunication between the space below the plunger and the space abovethe plunger was through a platelet receptor cavity. The float wascomprised of polyethylene and polycarbonate in such proportion as tohave an overall density of 1.08 g/ml. The outer diameter of the floatwas 2.535 cm and its overall length was 1.2 cm. The float was insertedinto a rigid polycarbonate tube with an inner diameter of 2.540 cm andlength 11.43 cm. The bottom of the rigid tube was sealed.

The device was filled with 25 cc of freshly drawn whole bloodanti-coagulated with CPDA-1. The device was centrifuged in an IEC CRU5000 centrifuge for 15 minutes at 1800 rpm. Following centrifugation,the plunger was depressed by inserting a blunt hypodermic needleconnected to a 10 cc syringe through the central access port andpressing down on the body of the syringe until it collided with the stopon the top of the float. As the syringe body was depressed, plateletpoor plasma collected in it. The syringe containing platelet poor plasmawas removed and a second syringe was attached to the needle. The devicewas swirled vigorously to resuspend the platelets within the plateletreceptor cavity after withdrawing 0.5 cc through the hypodermic needle(platelet extraction tube). An additional 3.5 cc concentrated plateletswas removed from the platelet receptor cavity through the hypodermicneedle (platelet extraction tube).

One half cc of this sample was diluted with 10 cc of Isoton II isotonicdiluent and centrifuged at 500 g for 1.5 minutes. One half cc of thisdiluted sample was diluted in yet another 10 cc of Isoton II andparticles larger than 3 fl counted on a Coulter Z-1 particle analyzer.This result was compared to the number of particles in a similarlytreated sample of whole blood. These small particles from treatedsamples represent the platelets. The sample of concentrated plateletscontained 74% of the platelets present in the introduced whole blood ata concentration 4.61 times that found in the whole blood. Since thisconcentration is much larger and the concentration of platements in theerythrocyte layer is much lower than obtained with simple centrifugationunder comparable conditions without the float, it is clear that the flowof erythrocyte suspension between the walls of the float and the tubeduring centrifugation gently disrupts the erythrocytes and releasesentrapped platelets, allowing them to collect in the platelet orbuffy-coat layer.

With the “Plunger” device without snorkel used in this example, theplatelet poor plasma is collected in a syringe during depression of theplunger. This provides all the advantages of “standard” plunger deviceplus providing platelet poor plasma in syringe for anyone who might wantto use it, for example, as a hemostat.

Various alternative configurations of the device are possible within thecontext of the present invention. For example, the two cones whichcomprise the buoy can be replaced by funnels or by cones possessingconcavities that communicate between the various compartments andconduct sedimenting cells between compartments during sedimentation.Complete fluid isolation of the various compartments is not essential,provided any openings between compartments are sufficiently small as toprevent substantial mixing of the fractions during handling andresuspension and withdrawal of the buffy coat. Means can be provided forrecovery of platelet depleted plasma and erythrocytes if desired. Thetube and the channel through which blood is introduced and the buffycoat is withdrawn need not be concentric or rigid. The elastomericsleeve can be replaced by a compressible material, e.g., foam, providedthe inner surface which contacts blood is smooth and does not trap oractivate platelets.

1-22. (canceled)
 23. A blood platelet separation device comprising: acentrifugal spin-separator container comprising a flexible tube havingan inner surface and a float positioned within the flexible tube; thefloat having a base and a platelet collection surface above the base,the platelet collection surface positioned on the float at a level whichplaces it immediately below the level of platelets after centrifugation,the float having a density less than the density of erythrocytes andgreater than the density of plasma; and the float having an outersurface engaging the inner surface of the flexible tube in a neutralpressure condition and configured to disengage from the inner surface ofthe flexible tube in an elevated pressure condition.
 24. The bloodplatelet separation device of claim 23 wherein the engagement betweenthe float and the outer surface of the float is configured to preventmovement of fluid between the outer surface of the float and the innersurface of the flexible tube in a neutral pressure condition.
 25. Theblood platelet separation device of claim 23 wherein the outer surfaceof the float disengages from contact with the inner surface of theflexible tube in the elevated pressure condition occurring duringcentrifugation, thus enabling movement of fluid between the outersurface of the float and the inner surface of the flexible tube andmovement of the float within the cylinder to a position placing theplatelet collection surface immediately below the level of platelets.26. A process for separating platelets from whole blood with a flexibleinner tube having an inner surface and a distal end; a float positionedwithin the flexible inner tube; the float having an outer surfaceengaging the inner surface of the flexible tube in a neutral pressurecondition, the engagement between the outer surface of the float and theinner surface of the flexible tube substantially preventing movement offluid between the outer surface of the float and the inner surface ofthe flexible tube; the float having a platelet collection surfacepositioned immediately below the level of the platelets in separatedblood after centrifugation; the process comprising the steps of a)introducing whole blood into the inner tube; b) subjecting the tube tocentrifugation forces in the axial direction toward the distal end,causing the outer surface of the float to disengage from the innersurface of the flexible tube, enabling movement of fluid between theouter surface of the float and the inner surface of the flexible tubeand movement of the float within the tube; and causing blood cells toconcentrate at the distal end, and platelets to collect at a levelimmediately above the platelet collection surface; and c) removingplatelets from the platelet collection surface.
 27. The process of claim26 wherein the fluid moves exclusively between the outer surface of thefloat and the inner surface of the flexible tube during centrifugation.28. A method of float movement in a blood platelet separation device,comprising: a) positioning a float with a platelet collection surfacewithin a flexible tube; b) neutralizing pressure in the flexible tubewith float, causing the outer surface of the float to engage an innersurface of the flexible tube and preventing substantial movement offluid between the outer surface of the float and the inner surface ofthe flexible tube; and c) elevating pressure in the flexible tube withfloat to create an elevated pressure condition during centrifugalseparation of platelets, causing the outer surface of the float todisengage from the inner surface of the flexible tube inner surface andenabling movement of fluid between the outer surface of the float andthe inner surface of the flexible tube, the float movement positioningthe platelet collection surface at a level immediately below the levelof platelets following centrifugal separation of platelets.
 29. A methodof float movement in a blood platelet separation device of claim 28wherein the fluid moves exclusively between the outer surface of thefloat and the inner surface of the flexible tube during centrifugalseparation of platelets. A formal claim listing is attached as aseparate document to meet the requirements of MPEP S1 121.